Production of chemicals from micro-organisms has been an important application of biotechnology. Typically, the steps in developing such a bio-production method may include 1) selection of a proper micro-organism host, 2) elimination of metabolic pathways leading to by-products, 3) deregulation of desired pathways at both enzyme activity level and the transcriptional level, and 4) overexpression of appropriate enzymes in the desired pathways. In preferred aspects, the present invention has employed combinations of the steps above to redirect carbon flow from phenylalanine through enzymes of the plant phenylpropanoid pathway which supplies the necessary precursor for the desired biosynthesis of pinosylvin.
Pinosylvin (or pinosylvine or 3,5-dihydroxy-trans-stilbene) is a phytophenol belonging to the group of stilbene phytoalexins, which are low-molecular-mass secondary metabolites that constitute the active defence mechanism in plants in response to infections or other stress-related events. Stilbene phytoalexins contain the stilbene skeleton (trans-1,2-diphenylethylene) as their common basic structure: that may be supplemented by addition of other groups as well (Hart and Shrimpton, 1979, Hart, 1981). Stilbenes have been found in certain trees (angio-sperms, gymnosperms), but also in some herbaceous plants (in species of the Myrtaceae, Vitaceae and Leguminosae families). Said compounds are toxic to pests, especially to fungi, bacteria and insects. Only few plants have the ability to synthesize stilbenes, or to produce them in an amount that provides them sufficient resistance to pests.
The synthesis of the basic stilbene skeleton is pursued by stilbene synthases, which comprises a small gene family in most species examined (Kodan et al. 2002). Stilbene synthases appear to have evolved from chalcone synthases, and belong to a polyketide synthase (PKS) superfamily that share more than 65% amino acid homology. Unlike the bacterial PKSs, both stilbene- and chalcone synthases function as unimodular PKSs with a single active site, forming relatively small homodimers (Tropf et al., 1995). Stilbene- and chalcone synthases use common substrates, three malonyl-CoAs and one cinnamoyl-CoA/p-coumaroyl-CoA, forming their products with similar reaction mechanisms (Kindl, 1985). Stilbene synthases can be classified into either a 4-coumaroyl-CoA-specific type that has its highest activity with 4-coumaroyl-CoA as substrate, such as resveratrol synthase (EC 2.3.1.95), or a cinnamoyl-CoA-specific type that has its highest activity with cinnamoyl-CoA as substrate, such as pinosylvin synthase (EC 2.3.1.146). Genes encoding resveratrol synthases have been described earlier for peanut (Arachis hypogaea) (Schöppner and Kindl, 1984; Schröder et al., 1988) and grapevine (Vitis vinifera) (Melchior and Kindl, 1991; Wiese et al., 1994) whereas genes encoding pinosylvin synthase have been mostly described for pine (Pinus sylvestris and -strobus) (Schanz et al., 1992; Raiber et al., 1995; Kodan et al., 2002; Hemingway et al., 1977).
Pinosylvin is present in the wood pulp of eucalyptus-, spruce- and pine trees such as Pinus sylvestris, -densiflora, -taeda and -strobus. In pine species, the constitutive pinosylvin occurs exclusively in the heartwood (Kindl, 1985). However, the compound is induced in the sapwood, phloem, and needles as a response to wounding, fungal attack or environmental stress such as UV-radiation and ozone exposure (Hart, 1981; Kindl, 1985; Richter and Wild, 1992; Lieutier et al., 1996; Rosemann et al., 1991). The compound possesses potent anti-fungal activity against a wide assortment of fungi (Lindberg et al., 2004; Pacher et al., 2002).
Pinosylvin (FIG. 1 trans-form) consists of two closely connected phenol rings and belongs therefore to the polyphenols. Unlike most other hydroxystilbenes, pinosylvin lacks a hydroxyl group in ring B (FIG. 1) and originates by condensation of unsubstituted cinnamoyl-CoA with three molecules of malonyl-CoA. That said, pinosylvin is structurally similar to the tri-hydroxystilbene resveratrol, which is found in red wine (Aggarwal et al., 2004). Much data has been generated demonstrating the health benefits of resveratrol. For instance resveratrol's potent anticancer activity across many cancer cell lines has well been established (Aggarwal et al., 2004). Given the similarity in structure with resveratrol, it is anticipated that pinosylvin possesses potent health benefits as well. Indeed pinosylvin's effect on various cancers, including colorectal- and liver cancers, has been studied, and has indicated it's chemopreventative- and anti-leukemic activity (Skinnider and Stoessl, 1986; Mellanen et al., 1996; Roupe et al., 2005 and 2006). Moreover, pinosylvin has anti-oxidant capacity as well, though to a lesser extent than, for instance, resveratrol (Stojanovic et al., 2001).
Presently, pinosylvin is mostly obtained in a mixture of various flavonoids that is extracted from the bark of pine. Said extraction is a labour intensive process with a low yield. In preferred aspects, the present invention provides novel, more efficient and high-yielding production processes.
In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, flavonoids and phytoalexins. Indeed formation of stilbenes in plants proceeds through the phenylpropanoid pathway. The amino acid L-phenylalanine is converted into trans-cinnamic acid through the non-oxidative deamination by L-phenylalanine ammonia lyase (PAL) (FIG. 2). From trans-cinnamic acid the pathway can branch into a resveratrol-forming route or into a pinosylvin forming route. In the first route trans-cinnamic acid is hydroxylated at the para-position to 4-coumaric acid (4-hydroxycinnamic acid) by cinnamate-4-hydroxylase (C4H), a cytochrome P450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450 reductase (CPR). Subsequently, 4-coumaric acid, is then activated to 4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). A resveratrol synthase (VST1), can then catalyze the condensation of a phenylpropane unit of 4-coumaroyl-CoA with malonyl CoA, resulting in formation of resveratrol. In the latter route trans-cinnamic acid is directly activated to cinnamoyl-CoA by the action of 4CL where a pinosylvin synthase (PST) subsequently catalyzes the condensation of a phenylpropane unit of cinnamoyl-CoA with malonyl CoA, resulting in formation of pinosylvin.
Stilbene synthases are rather promiscuous enzymes that can accept a variety of physiological and non-physiological substrates. For instance, addition of various phenylpropanoid CoA starter esters led to formation of several products in vitro (Ikuro et al., 2004; Morita et al., 2001). Likewise it has been shown that resveratrol synthase from rhubarb (Rheum tartaricum) indeed synthesized a small amount of pinosylvin when cinnamoyl-CoA was used as substrate instead of coumaroyl-CoA (Samappito et al., 2003).
Similarly, coumaroyl-CoA ligase can accept both coumaric acid and cinnamic acid as substrate, albeit with a catalytic efficiency (Km/Kcat) that is 100 times less for cinnamic acid compared to coumaric acid (Allina et al., 1998; Ehlting et al., 1999). We deduced from the above that it would be possible to produce pinosylvin in a pathway that would consist of a 4CL and a stilbene synthase, even one that is designated as a classical resveratrol synthase.
Recently, a yeast was disclosed that could produce resveratrol from coumaric acid that is found in small quantities in grape must (Becker et al. 2003, ZA200408194). The production of 4-coumaroyl-CoA from exogenous 4-coumaric acid, and concomitant resveratrol, in laboratory strains of S. cerevisiae, was achieved by co-expressing a heterologous coenzyme-A ligase gene, from hybrid poplar, together with the grapevine resveratrol synthase gene (VST1). The other substrate for resveratrol synthase, malonyl-CoA, is already endogenously produced in yeast and is involved in de novo fatty-acid biosynthesis. The study showed that cells of S. cerevisiae could produce minute amounts of resveratrol, either in the free form or in the glucoside-bound form, when cultured in synthetic media that was supplemented with 4-coumaric acid.
Given the promiscuity of the resveratrol synthase, it may be that said yeast could produce pinosylvin as well when fed with substantial amounts of cinnamic acid. However, commercial application of such a yeast would be hampered by the probable low pinosylvin yield, and the need for addition of cinnamic acid, which is not abundantly present in industrial media. Hence, to accelerate and broaden the application of pinosylvin as both a pharmaceutical and neutraceutical, it is highly desirable to provide a yeast or other micro-organism that can produce pinosylvin directly from glucose, without addition of cinnamic acid or any downstream cinnamic acid derivative such as cinnamoyl-CoA.
A recent study (Ro and Douglas, 2004) describes the reconstitution of the entry point of the phenylpropanoid pathway in S. cerevisiae by introducing PAL, C4H and CPR from Poplar. The purpose was to evaluate whether multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. By feeding the recombinant yeast with [3H]-phenylalanine it was found that the majority of metabolized [3H]-phenylalanine was incorporated into 4-[3H]-coumaric acid, and that phenylalanine metabolism was highly reduced by inhibiting C4H activity. Moreover, PAL-alone expressers metabolized very little phenylalanine into cinnamic acid. When feeding [3H]-phenylalanine and [14C]-trans-cinnamic acid simultaneously to the triple expressers, no evidence was found for channeling of the endogenously synthesized [3H]-trans-cinnamic acid into 4-coumaric acid. Therefore, efficient carbon flux from phenylalanine to 4-coumaric acid via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and sheer biochemical coupling of PAL and C4H seems to be sufficient to drive carbon flux into the phenylpropanoid pathway. In yet another study (Hwang et al., 2003) production of plant-specific flavanones by Escherichia coli was achieved through expression of an artificial gene cluster that contained three genes of a phenyl propanoid pathway of various heterologous origins; PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor, and chalcone synthase (CHS) from the licorice plant Glycyrrhiza echinata. These pathways bypassed C4H, because the bacterial 4CL enzyme ligated coenzyme A to both trans-cinnamic acid and 4-coumaric acid. In addition, the PAL from Rhodotorula rubra uses both phenylalanine and tyrosine as the substrates. Therefore, E. coli cells containing the gene clusters and grown on glucose, produced small amounts of two flavanones, pinocembrin (0.29 g/l) from phenylalanine and naringenin (0.17 g/l) from tyrosine. In addition, large amounts of their precursors, 4-coumaric acid and trans-cinnamic acid (0.47 and 1.23 mg/liter respectively), were accumulated. Moreover, the yields of these compounds could be increased by addition of phenylalanine and tyrosine.
Also described are studies in which the enzyme properties of pinosylvin synthases are studied by first cloning the genes into Escherichia coli. For instance, Raiber et al., 1995 report on stilbenes from Pinus strobus (Eastern white pine) that were investigated after heterologous expression in Escherichia coli. For this a P. strobus cDNA library was screened with a stilbene synthase (STS) probe from Pinus sylvestris and amongst the isolated cDNAs two closely related STS genes, STS1 and STS2, were found with five amino acid differences in the proteins. The genes were cloned on a plasmid and expressed into E. coli, and cell extracts were subjected to enzyme assays. It appeared that both proteins accepted cinnamoyl-CoA as a substrate and thus were considered as pinosylvin synthases, however they revealed large differences. STSI had only 3-5% of the activity of STS2, and its pH optimum was shifted to lower values (pH 6), and it synthesized with cinnamoyl-CoA a second unknown product. Site-directed mutagenesis demonstrated that a single Arg-to-His exchange in STS1 was responsible for all of the differences. In another study three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) from Pinus densiflora were isolated and the cDNAs were heterologously expressed in E. coli to characterize their enzymatic properties (Kodan et al., 2002). PDSTS3 appeared to be an unusual STS isozyme that showed the highest pinosylvin-forming activity among the STSs tested. Furthermore, PDSTS3 was insensitive to product inhibition unlike PDSTS1 and PDSTS2. The unusual characteristics of PDSTS3 could be ascribed to a lack of a C-terminal extension that normally is common to stilbene synthases, which was caused by a frame-shift mutation. In yet another study a genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe (Müller et al., 1999). After subcloning, four different members were characterized by sequencing. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%.
Differences in promoter strength were then analysed by transient expression in tobacco protoplasts. Constructs used contained the bacterial-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.
A further study showed that a stilbene synthase cloned from Scots pine (Pinus sylvestris) was earlier abortively assigned as a dihydropinosylvin synthase, while it showed to be a pinosylvin synthase. The previous mis-interpretation was caused by the influence of bacterial factors on the substrate preference and the activity of the plant-specific protein that was expressed in E. coli. After improvement of the expression system, the subsequent kinetic analysis revealed that cinnamoyl-CoA rather than phenylpropionyl-CoA was the preferred substrate of the cloned stilbene synthase. Furthermore, extracts from P. sylvestris contained factor(s) that selectively influenced the substrate preference, i.e. the activity was reduced with phenylpropionyl-CoA, but not with cinnamoyl-CoA. This explained the apparent differences between plant extracts and the cloned enzyme expressed in E. coli and cautions that factors in the natural and the new hosts may complicate the functional identification of cloned sequences.
Furthermore, vectors are described with stilbene synthase genes, which can mean resveratrol synthase and pinosylvin synthase, for the transformation of organisms and plants to confer enhanced resistance against pests and wounding (EP0309862 and EP0464461).
Also, further vectors are described that contain DNA sequences that will hybridise to pinosylvin synthase of Pinus sylvestris (U.S. Pat. No. 5,391,724) and said vectors to be used for expression in a plant (U.S. Pat. No. 5,973,230). The incorporation of PAL and 4CL together with a stilbene synthase for the production of pinosylvin in a organism is not however disclosed. Nor are any pinosylvin producing micro-organisms.
Recently, evidence was shown that the filamentous fungi A. oryzae contained the enzyme chalcone synthase (CHS) that is normally involved in the biosynthesis of flavonoids, such as naringenin, in plants (Juvvadi et al., 2005; Seshime et al., 2005). Indeed it was also shown that A. oryzae contained the major set of genes responsible for phenylpropanoid-flavonoid metabolism, i.e PAL, C4H and 4CL. However, there is no evidence that A. oryzae contains a stilbene synthase.
Our co-pending application WO2006/089898 describes resveratrol producing micro-organisms, especially yeasts.